Methods for high scale therapeutic production of memory NK cells

1. A method for increasing the number of memory Natural Killer (NK) cells comprising a) preactivating NK cells by contacting at least one NK cell with at least one or more stimulatory cytokines; and b) expanding the preactivated NK cells of step a) by contacting said cells with plasma membrane (PM) particles with surface bound interleukin (IL)-21 (PM21) particles, exosomes (EX) with surface bound IL-21 (EX21) exosomes, or feeder cells (FC) with surface bound IL-21 (FC21) feeder cells; wherein the NK cells are further contacted with stimulatory peptides 4-1BB ligand (4-1BBL) and IL-21.

2. The method of claim 1 , wherein the NK cells are obtained from an unselected population of peripheral blood mononuclear cells.

3. The method of claim 1 , wherein the at least one or more stimulatory cytokines is selected from the group consisting of Interleukin-12 (IL-12), Interleukin-15 (IL-15), and Interleukin-18 (IL-18).

4. The method of claim 3 , wherein the method comprises contacting the NK cell with 3 stimulatory cytokines.

5. The method of claim 3 , further comprising contacting the NK cell with a stimulatory peptide selected from the group consisting of Interleukin-2 (IL-2), Major histocompatibility complex class I-related chain A/B (MICA/B), UL16 Binding Protein 2 (ULBP2), Intracellular adhesion molecule 1 (ICAM-1), 2B4, /signaling lymphocyte activation molecule (SLAM) family member 2 (F2) (BCM1/SLAMF2), cluster of differentiation 155 (CD155), cluster of differentiation 112 (CD112), C-C chemokine receptor type 7 (CCR7), DnaX activation protein of 12 kDa (DAP12), and DnaX activation protein of 10 kDa (DAP10).

6. The method of claim 1 , wherein the NK cells are contacted with the IL-12, IL-15, or IL-18 in vitro, in vivo, or ex vivo.

7. The method of claim 1 , wherein the NK cells are contacted with the one or more stimulatory cytokines for between about 6 to about 24 hours.

8. The method of claim 1 , wherein the PM21 particles, EX21 exosomes, or FC21 feeder cells further comprise one or more stimulatory peptides coupled to a membrane-inserting peptide.

9. The method of claim 8 , wherein the membrane-inserting peptide comprises a fused peptide that is capable of membrane insertion, with affinity for a lipid bilayer, and wherein said fused peptide comprises a segment of IG4, cluster of differentiation 4 (CD4), or a combination thereof.

10. The method of claim 8 , wherein the one or more stimulatory peptides coupled to a membrane-inserting peptide is a fusion protein encoded by recombinant deoxyribonucleic acid (DNA).

11. The method of claim 8 , wherein the membrane-inserting peptide comprises human fragment crystallizable region (Fc), Glycosylphosphatidylinositol (GPI), trans-membrane T-cell receptor, or pH low insertion peptide (pHLIP).

12. The method of claim 8 , wherein the one or more stimulatory peptides are selected from the group consisting of IL-2, IL-18, IL-21, MICA/B, ULBP2, ICAM-1, 2B4, BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10.

13. The method of claim 1 , wherein the NK cells are contacted with the PM21 particles, EX21 exosomes, or FC21 feeder cells in vitro, in vivo, or ex vivo.

14. The method of claim 1 , wherein the NK cells of step a are contacted with PM21 particles, EX21 exosomes, or FC21 feeder cells for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days.

15. The method of claim 1 , further comprising resting the expanded memory NK cells following step b).

16. The method of claim 15 , wherein the memory NK cells are rested for at least 1, 2, 3, 4, or 5 days.

17. The method of claim 15 , wherein the memory NK cells are rested for no more than 1, 2, 3, 4 or 5 days.

18. The method of claim 1 , wherein the PM21 particles, EX21 exosomes, or FC21 feeder cells comprise 4-1BBL and IL-21.

19. The method of claim 18 , wherein the 4-1BBL and IL-21 are coupled to a membrane-inserting peptide.